wjd

WJD

10.5005/jp-journals-10015-1495
Troubleshooters in Light Microscopy
Review Article

Troubleshooters in Light Microscopy

1
A Lavanya, 2SV Sowmya, 3Roopa S Rao, 4Dominic Augustine, 5Vanishri C Haragannavar, 6Shwetha Nambiar

ABSTRACT a huge challenge. Although currently available diag-

The aim of this study is to review the significance of parts nostic and prognostic techniques are sophisticated and
and operating principle of compound light microscopy with advanced, pathologists have to rely on light microscopes
emphasis on their troubleshooters. Light microscope has even today. The microscope is derived from a Greek word
been an important diagnostic tool in scientific research over wherein “mikro” means “small” and “skope” means
the years. The invention of the microscope by Anthony Leeu-
wenhoek has given rise to an interesting dimension in life
“to look.” This optical instrument consists of a lens for
sciences where great discoveries have been made to study achieving a clear and magnified image of an object, i.e.,
various microorganisms and the structure of cells. Handling the not visible to the naked eye. The use of microscopes was
microscope requires sound knowledge about its parts and their initiated by the Romans who employed glasses instead
construction for effective functioning. There are innumerable
of lenses. Then, with a discovery that an image could be
faults regardless of the elegance of the microscope equipment
or the experience level and skill of the user. Errors must be obtained when sun rays were focused on special glasses,
addressed carefully to detect the cause that may be due to called magnifiers or burning glasses, made a differ-
poor specimen preparation or technical issues. The majority ence in the field of pathology. In 1609, Galileo Galilei
of photomicrography errors traced on the optical configuration developed a compound microscope with a convex and
of the microscope include improper illumination, use of the
wrong filters, incorrect setting of the substage components and
concave lens.1
preservation, processing and mounting of tissue specimens, Scientists have a variety of optical instruments to
or microscopic optics. By capturing various photo­micrographic perform routine laboratory procedures and research, but
images at different erronic modes of microscopic handling and the compound microscope is the “workhorse” and com-
artifactual appearances, the acceptable approach for the use
monly found in laboratories. The microscopes introduced
of compound microscope has been emphasized with possible
remedies to overcome them. Comprehension of the compound so far produce a magnified image of the specimen and
light microscopic components with causes and remedies of render the details visible to the human eye or camera. It is
troubleshooters may lead to efficient handling of the instrument important to know the relation between the eyes and micro-
for investigative and diagnostic purposes. scope. For an image to be viewed, it must be presented to the
Clinical significance: Good knowledge of the right usage of eye in colors (visible spectrum) and with varying degrees of
microscope parts is pivotal in routine laboratory investigations light intensity. Although we maintain a good relationship
for the diagnosis and prognosis of pathologies and scientific
between the eyes and microscope, there are other different
research.
sources that can hamper good image formation.2
Keywords: Light, Microscopy, Troubleshooters. A sound knowledge of the construction and limitations
How to cite this article: Lavanya A, Sowmya SV, Rao RS, of the microscope is important for its efficient usage. An ill-
Augustine D, Haragannavar VC, Nambiar S. Troubleshooters adjusted, badly illuminated microscope can give completely
in Light Microscopy. World J Dent 2017;8(6):511-518.
misleading information about a particular disease.1,3 There
Source of support: Nil are different sources of error in setting up a microscope, and
Conflict of interest: None it is not easy to track their precise cause. Hence, for better
diagnosis, it is important to know the construction and
functions of each part to eliminate their limitations. This
INTRODUCTION
review focuses on the importance of appropriate usage of
The present lifestyle and environmental changes have microscope parts and their troubleshooters.
paved way to new diseases, and their diagnosis poses
PRINCIPLE OF MICROSCOPE
A compound microscope is an optical instrument consist-
1-6
Department of Oral Pathology and Microbiology, Faculty of
ing of two convex lenses which are used for magnifying
Dental Sciences, M. S. Ramaiah University of Applied Sciences
Bengaluru, Karnataka, India very small objects. The two series of lenses are the objec-
tive lens and the eyepiece lens. The lens placed toward
Corresponding Author: A Lavanya, Department of Oral
Pathology and Microbiology, Faculty of Dental Sciences, M. S. the object is represented as an objective lens, and the one
Ramaiah University of Applied Sciences, Bengaluru, Karnataka closer to our eyes is eyepiece lens. The focal length of the
India, Phone: +918610702985, e-mail: drlavsmdsop92@gmail. objective lens is shorter than the eyepiece lens as it helps
com
in receiving more light rays from an object and forms a
World Journal of Dentistry, November-December 2017;8(6):511-518 511
A Lavanya et al

bright image. Many compound microscopes are binocular

and have two ocular lenses. A binocular type will have
a prism, either in the head or in the body tube that helps
to split the image and direct it to the eyepiece.1,4

WORKING PRINCIPLE
When light from the light source is passed through a
thin transparent object, condensation of rays is brought
about by the substage condenser through the numerical
aperture. There is a substage condenser located between
the stage and light source that helps in condensing the
light rays. This type of light condensing and gathering
capacity of the condenser is called the numerical aperture
of the condenser. The first lens, called objective lens, col-
Fig. 1: Schematic diagram showing principle of compound light
lects the light passing through the object from the light microscope
source and then focuses it on forming a real image of an
object inside the microscope. Then, the formed image is
magnified by a second lens called as eyepiece lens and OPTICAL PARTS
is perceived as “virtual image” by the observer. As the Light Source
light passes directly from the source to the eye, wherein
The source is connected to the mains through a regula-
the field of vision is brightly illuminated, it is also called
bright-field microscope (Fig. 1).1,4,5 tor that controls the brightness of the field. Choosing
the appropriate light source for investigation in optical
FUNCTIONAL SIGNIFICANCE OF PARTS microscopy highly depends on the illumination strategy,
OF COMPOUND MICROSCOPE specimen parameters, microscope configuration, and
detector sensitivity.1,4
Compound microscope is divided into three basic
The causes for improper image formation due to the
structural components. They are body, base, and arm.
inadequate light source are a faulty connection of alternat-
The body contains the optical parts, and base helps in
ing current (AC) power cord, inoperative outlet, burned-
supporting the microscope and contains the illuminator,
out lamp, and blown fuse (Fig. 4B). This can be solved by
whereas arm acts as the connection between body and
the use of appropriate outlet connection, having qualified
head. It has also been categorized mainly into optical and
service and repair with the replacement of lamp and fuse.6,7
mechanical parts based on their function.
• Optical parts include light source, diaphragm, filter,
Diaphragm
condenser, objective, and eyepiece (Fig. 2C).
• Mechanical parts include base, milled knobs, mechani­ The diaphragm is placed below the stage. It is used to
cal stage, rack stop, curved arm, nose piece, beam control the amount of light reaching the specimen and
splitter (Fig. 3C), draw and body tubes. angle of cone entering the objective.1 The depth of the
When all these microscopic parts function effectively, field, numerical aperture, and image quality are affected
the image obtained will be of superior quality (Fig. 4A). by the opening or closure of the aperture diaphragm.1,4

A B C
Figs 2A to C: Parts of the compound light microscope: (A) Eyepiece with diopter adjustment; (B) objectives with color coding,
red 4× (m), yellow 10× (n), blue 40× (p), white 100× (q); and (C) optical parts of microscope

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Troubleshooters in Light Microscopy

B C
Figs 3A to C: (A) Photograph showing adjusting knobs; (B) safety rack stop;
and (C) mechanical parts

The main aim is to achieve a balance between the resolu- component that collects light from the light source and
tion and contrast. If the aperture diaphragm is opened focuses it on the specimen.1 Abbe, aplanatic, and achro-
wide, the result will be a washed-out image with no con- matic are the three types of condensers. It provides sharper
trast and difficulty in obscuring the details of the image. and clearer image and can be raised or lowered by turning
If there is over closure of aperture diaphragm, grainy the milled knobs/adjusting knobs. Most widely used is
image with less resolution will be obtained (Fig. 4C).6-9 the Abbe condenser that produces a perfect image when a
condenser with a lens system equal to that of the objective
Filter is selected.1,5,6
Filters are placed in the path of light and help in increas- Unevenly illuminated objective field with poor image
ing the contrast, blocking the ambient light, decreasing quality is formed when the condenser is being inappro-
the intensity, and absorbing excess heat. A lamp with priately lowered or moved away from the stage (Fig. 4E).
tungsten filament has a tendency to change their color Clear image with good intensity may be obtained by
and temperature depending on the light intensities. A realigning the condenser and matching with the objec-
filter placed over the lamp absorbs the red part of the tive being used.6,7,9
spectrum and gives more neutral color.
There are various types of filters, such as neutral
Objective Lens
density, colored, color correcting, heat absorbing, and
exciter filter.1 Objective lenses are those that are closer to the object. A
Neutral filters are used in decreasing the brilliance of compound microscope generally has four objective lenses
illumination, i.e., most commonly used in photomicro­ with different magnifications and twined into a circular
graphy. If neutral density filter is not inserted into the light nose piece which may be rotated to select the desired
pathway, it results in increased intensity of light. This leads magnification. The objective lenses are 4×, 10×, 40×, and
to color temperature imbalance between the light source 100× which are color-coded as red, yellow, blue, and white
and the film emulsion that produces an unexpected color respectively, for rapid identification (Fig. 2B).1,4,5 Table 1
shift.1,4 If the color temperature of the light source is too summarizes the available objective lenses with their
low, orange-yellow-colored image is observed (Fig. 4D). respective color codes.
This can be corrected by inserting the neutral density Objective lenses are mainly of two types—achromatic
filters which provide uniform intensity and balanced and apochromatic that are responsible for magnifying the
color temperature over the entire microscopic field view.6,7 image of a specimen. The different colors of light while
passing through the objective lens result in an image
Condenser Lens having colored fringes around them due to differences
The condenser may be movable or fixed. When moved, it in wavelength. This effect can be prevented using ach-
shifts in horizontal and vertical direction. It is a substage romatic and apochromatic objectives.1,4
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A Lavanya et al

A B C D

E F G H
Figs 4A to H: Photomicrographs of hematoxylin and eosin-stained tissues showing light microscopic
operating errors (×100): (A) Optimal architectural and cellular details with ideal microscope
construction; (B) light background without image; (C) grainy image by overclosure of the diaphragm;
(D) orange-yellow-colored image; (E) unevenly illuminated image; (F) enhanced cellular features with
oil immersion (×1,000); (G) unsharp image without oil immersion (×1,000); and (H) spotty image (×100).

Achromatic objectives are used for correcting the lens observer’s eye called “eye lens.” These two lenses together
error of chromatic aberrations at two wavelengths—red are known as “ocular lenses” which have a power of
and blue. Apochromatic objectives are made up of several magnification of about 10 times that of an objective and
fluorite lenses with glass lens and used for correction of helps in magnifying the real image formed by the object
chromatic aberration at three wavelengths—red, blue, (Fig. 2A). The eyepiece is the easiest component to rotate
and green.1,4 and get grotty in normal use.1,10
Dark spots and poor resolution in the focused image There are various types of eyepiece lenses which are
are due to the dirt or contamination with dust particles • Huygens—negative undercorrected and used with
of the objective lens. This can be corrected by cleaning achromatic objectives
the lenses with lens paper.6,7,9 • Ramsden—positive and used for micrometry
• Widefield—provides large flat view
Oil Immersion Objective • High eyepoint—used by microscopists who wear
Oil immersion is designed specifically to have the same spectacles
refractive index as glass, and as a result, there is no • Compensatory—used with apochromatic objectives
bending of light between the objective and specimen. Oil The veiled and spotty images produced may be due
immersion is used for viewing the bacteria, blood cells, to dirt or oil on the eyepiece which can be identified by
and striations in skeletal muscle (Fig. 4F). twir­ling the eyepiece (Fig. 4H). This can be corrected by
Hazy, unsharp, and poor resolution images are due cleaning the eyepiece with 70% isopropyl alcohol with
to the use of 100× objective without oil, contamination of lens paper.6,7
bottles containing cedarwood oil, and wrong immersion
media (Fig. 4G). This can be prevented by placing the Diopter Setting
correct immersion medium without contamination. Clog- For optimal visualization, there are three main steps to
ging of the objective lens with oil can be cleaned using lens be followed:
paper, and use of xylene should be avoided as it damages 1. First, adjust the interpupillary distance and also
the lens.6,7 binocular vision until the right and left fields of
view concise completely. Record the value for future
Eyepiece
adjustment.1
An eyepiece consists of two convex lenses: One facing 2. Second, a control knob called diopter which is present
the objective called “field lens” and other placed near the only in the left eyepiece is adjusted. By looking

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Table 1: Objective lenses with their color coding Milled Knobs

Objective lens Color code
There are two types of knobs—coarse- and fine-adjustment
5× Red
ones (Fig. 3A). Coarse adjustment is used to adjust the slide
10× Yellow
16× Green containing the specimen in back and forth direction to
20× Green achieve a focused image. Fine adjustment helps to bring
25× Turquoise the specimen into the sharper focus.1,4
32× Turquoise The unfocused and unsharp image occurs due to
40× Light blue over tightening of the coarse adjusting knob as tension
50× Light blue is extremely high (Fig. 6A). This can be prevented by
60× Cobalt blue loosening the ring properly for refocusing the image. If
63× Cobalt blue
the focus obtained is lost immediately, it may be due to
100× White
light tension. This can be corrected by rotating the knob
150× White
250× White
clockwise to increase the tension to obtain the focused
Oil immersion Color code image.6,7,9
Cedarwood oil Black
Glycerol Orange Mechanical Stage
Water White A mechanical stage is a flat surface where the slide with
the specimen is placed and moved in a side-to-side direc-
through the right eyepiece, first adjust the adjusting tion. It is fitted with Vernier scale with readings from
knobs to bring the specimen into focus. 100 to 170 mm for recording the position of the slide in
each direction. It is useful for repositioning of the slide
3. Third, look into the left eyepiece and adjust the diopter
at a later date for viewing a specific structure.1,4
ring to bring a fine focus (Fig. 2A).1
Eye fatigue results when there is incorrect diopter
Rack Stop
adjustment. This can be solved by correctly adjusting
the diopter focus. Two separate or blurred images appear Safety rack stop is an adjustable screw situated between
due to the incorrect interpupillary distance between the the stage and the arm (Fig. 3B), which prevents the stage
eyepieces and are corrected by adjusting the eyepieces from coming too far and ramming against the objective
(Fig. 5).6,7,9 lens.11,12 Improper vertical adjustment may lead to the
formation of a light background or no image wherein the
body of the microscope is either far away or too close to
MECHANICAL PARTS
the object (Fig. 6B). This can be corrected by proper adjust-
Base ment of the rack stop and by maintaining the required
distance between the lens and the object.9
Base of the microscope is usually made up of durable
material as it supports the microscope to stand and Curved Arm
provides stability.1,4
The part of the microscope that holds the stage, body tube,
and adjusting knobs and helps in carrying the microscope
easily is the curved arm.1

Nosepiece/Turret
Nosepiece is the microscopic part that holds the objective
lenses to view the specimen in different magnifications.1
The partially illuminated objective field may occur due
to the improper clicking of the revolving nosepiece
(Fig. 6C). This can be solved by rotating the nosepiece till
it gets clicked into its position.6,7,9

Beam Splitter
It is an optical device that splits a beam of light. Improper
positioning of beam splitter results in partially illumi-
Fig. 5: Schematic diagram with two separate images of nated object field which is due to horizontal malalignment
hematoxylin and eosin-stained tissue sections (×100) (Fig. 6D).9
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A Lavanya et al

Draw Tube Coverslip

It is the upper part of body tube slightly narrower and holds A coverslip or cover glass is a thin flat piece of transparent
the eyepiece without getting slipped away during observa- material which is square or rectangular in shape of about
tion. It enables to adjust the mechanical tube length.1 0.17 mm thick and measures 25 × 75 mm. Coverslips
are available with variable lengths and thickness. It is
Body Tube placed over the specimen in the slide which prevents
The mechanical body tube length is the distance from the drying out of specimen and protects from bacterial
nosepiece opening where objective lenses are mounted contamination.1,4
till the top of observation tube where eyepieces are Cloudy image is formed due to the increased thick-
inserted. The accepted standardized body tube length is ness of the coverslip that results in higher numerical
160 mm (6.3 inches). When the objective and tube length aperture and loss of resolution (Fig. 6G). This can be
are mismatched, ghost images are produced that are corrected using type I coverslip for mounting. Misty
caused by converging light passing through the inclined and dark speck-like structures appear when mounting
plane glass surfaces. This can be corrected by maintaining media are placed over the coverslip (Fig. 6H). This can
optimal tube length.1 be prevented by proper placement of mounting media
and removing any excess medium that has flown over
ACCESSORY COMPONENTS OF MICROSCOPE the slide or coverslip.9

Slide
Tissue Thickness
The microscopic slide is a thin flat piece of glass that
The specimen thickness ranges from 3 to 5 µm.1 Due to the
measures 75 × 26 × 1 mm thick. It is used to hold the
increased thickness of the specimen, the image formed
specimen for microscopic examination.1 Unfocused and
appears distorted (Fig. 6I). This can be corrected using
steaminess of image are due to the placement of slide in
appropriate tissue thickness (4 µm).9
upside-down position (Fig. 6E). This can be corrected by
inverting the slide and viewing from the correct side.9
Mounting Media
Due to dust particles over the slide, dirty specks in the
field of view can be observed which will interfere during Mounting media is the liquid, resinous, water-soluble
examination and it moves when the slide is moved side- solution in which specimen is embedded under the cover
ways (Fig. 6F). This can be corrected by cleaning the slide glass. Most commonly used mounting media are dibutyl
with soft sterile cloth.9 pthalate polystyrene xylene, and its main function is

A B C D E

F G H I J
Figs 6A to J: Photomicrographs of hematoxylin and eosin-stained tissues showing light microscopic operating
errors (×100): (A) Unsharp image; (B) hazy image due to improper safety rack stop; (C) partly illuminated image;
(D) partially illuminated object field; (E) unfocused image due to improper slide placement; (F) dirty specks in field;
(G) cloudy image; (H) speck-like structures caused by mounting media on coverslip; (I) distorted image; and (J)
ground section showing air bubbles interfering visibility of tissues due to evaporation of xylene in mounting media

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to protect the tissue. Ideally, the refractive index of the Air bubbles will appear when mounting media with
mounting media should be as close as to the tissue, which evaporated xylene is used for the purpose of mounting.
is approximately 1.53, that induces a certain amount of This can be prevented using a proper mounting media
transparency and allows the stained tissue elements to placed in an air-tight container where xylene evaporation
be visible under microscope.13 is avoided (Fig. 6J).
When tissue section is placed without the mounting
media, the image produced will be of poor clarity and SUMMARY
contrast where the refractive index will be 1.0. This can The various troubleshooters during the use of the light
be corrected by placing the tissue with proper mounting microscope, their causes, and remedies have been sum-
media.13 marized in Table 2.

Table 2: Summary of troubleshooters, causes, and remedies in the usage of light microscope
Parts of
microscope Problems Causes Remedies
Eyepiece Veiled and spotty image Dirt or oil on the eyepiece, identified by twirling Use 70% isopropyl alcohol and lens
the eyepiece paper for cleaning the eyepiece
Eye fatigue Incorrect right and left diopter adjustment Correct adjustment of the diopter focus
Two separate or blurry Incorrect interpupillary distance between the Correct adjustment of the eyepieces
images eyepieces
Adjusting Unfocused and unsharp Increased tension due to overtightening of the Loosen the ring properly for refocusing
knobs image adjusting knobs the image
Focus obtained is lost Decreased tension due to too much tightness Rotating the knob clockwise to increase
of adjusting knobs the tension to obtain focused image
Nosepiece Partially illuminated Revolving nosepiece has not been clicked to Rotating the nosepiece till it gets clicked
(or) turret objective field its position into its position
Safety rack Dark background or no Improper vertical adjustment in which the body Proper adjustment of the rack stop helps
stop image of the microscope is either far away or too in maintaining the required distance
close to the object between the lens and the object
Beam An object field partially Horizontal maladjustment created by improper Placement of the beam splitter in the
splitter illuminated positioning of beam splitter proper position
Objective Field of view cut off Maladjustment of objective lens in which it is Positioning of objective lenses till it gets
lens not clicked into the position locked
Dark spots and poor reso- Dirt or contamination particles in the objective Cleaning the lenses using 70% isopropyl
lution in the focused image lens alcohol with lens paper
Oil Hazy, poor resolution with Without oil placed in 100× objective, Correct placement of immersion medium
immersion unsharp focused image contamination of bottles containing cedarwood without contamination
oil and wrong immersion medium
Microscopic Unfocused and Slide placed in the upside-down manner Inversion of the slide
slide steaminess of image
Dirty specks in the field Dust particles over the slide Cleaning the slide with soft sterile cloth
of view
Coverslip Cloudiness of image Increased thickness of the coverslip more than Use type I coverslip for mounting
0.17 mm
Mistiness and dark specks Mounting medium is placed over the coverslip Proper placement of mounting media
like structures
Tissue Distorted image Increased thickness of specimen Minimum thickness of 4 µm to be
thickness obtained
Condenser Unevenly illuminated Condenser lowered or moved away from the Realigning the condenser
lens object field with poor stage
image quality
Diaphragm Grainy image and less Overclosure of aperture diaphragm Proper positioning of diaphragm
resolution
Washed-out image and Aperture diaphragm is opened wide
difficult to obscure the
detail
Filter Orange-yellow-colored If neutral density filter is not inserted into the Insertion of the neutral density filters
image is obtained light pathway, it results in increased intensity of which provide uniform intensity and
light. This leads to color temperature imbalance balanced color temperature
between light source and film emulsion which
produces unexpected color shift
Light source No image AC power cord not connected, outlet Connecting the outlet plug properly,
inoperative, lamp burned out, fuse blown have qualified service repair, replace
lamp and fuse

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A Lavanya et al

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